梨磷脂酰胆碱转移蛋白基因的克隆及其表达分析

Molecular cloning and expression analysis of phosphatidylcholine transfer protein gene in pear

据转录组测序结果,结合RACE(cDNA末端快速扩增技术)和Genome walking(染色体步移技术)获得了梨磷脂酰胆碱转移蛋白基因的完整读码框序列,其开放阅读框长为1 323 bp,编码的440个氨基酸序列与葡萄磷脂酰胆碱转移蛋白质的同源相似性达73%,其具有START domain(启动域)、ZnF_TAZ(TAZ锌指结构)、DCD(双氰胺)保守结构域,将梨磷脂酰胆碱转移蛋白基因命名为PePCTP1。PePCTP1基因与GFP融合,构建植物表达载体,洋葱表皮亚细胞定位观察结果显示,PePCTP1基因编码的蛋白质分布在细胞膜上。用激素生长素(IAA)、脱落酸(ABA)、水杨酸(SA)、赤霉素(GA)、细胞分裂素(6-BA)及NaCl诱导处理,发现与对照(CK)相比,SA和GA处理后PePCTP1基因表达量显著上升;NaCl和IAA处理后PePCTP1基因的表达量随时间逐渐上升;而ABA和6-BA处理不能增加目的基因的表达量。据此推测PePCTP1可能参与梨的逆境胁迫抗性反应。

According to the results of transcriptome sequencing, the open reading frame of a phosphatidylcholine transfer protein gene （named as PePCTP1 ） was cloned from pear with a coding region of I 323 bp by RACE（ Rapid-ampli- fication of cDNA ends） and Genome walking, encoding a protein of 440 amino acid which was 73% identical to the phos- phatidylcholine transfer protein in grape. START domain, ZnF_TAZ and DCD conserved domains were found in PePCTP1. A plant expression vector was constructed by injecting PePCTP1 into a vector containing GFP open reading frame. Onions epidermal subceUular localization showed that PePCTP1 was located on the ceU membrane. The PePCTP1 gene expression pattern after IAA, ABA, SA, GA, 6-BA and NaCl treatments was compared with the control （CK）. Expression level of PePCTP1 increased significantly after treated with SA and GA, and increased gradually after treated with NaC1 and IAA. However,PePCTP1 showed no response to ABA and 6-BA treatments. It was suggested that PePCTP1 might be involved in the response to anti-stress of SA, GA, NaCl and IAA.